Diploid oyster spat were purchased from the Auburn University Shellfish Laboratory and settled onto 4.5 cm × 4.5 cm marble tiles. For one week after settlement, spat on tiles were caged and kept in 1250 L mesocosms with natural flowing seawater from Mobile Bay at a flow rate of 20 L/min. We then ensured each tile had at least 15 oyster spat and used high-density polyethylene fishing line to tie tiles back-to-back. Four tile pairs were placed in each aquarium, ensuring that every tile pair was upright to maintain good water flow around the spat. An intact, sun-bleached adult oyster shell was also placed into each aquarium for spat tile pairs to lean on so that they maintained an upright position. Aquaria were filled with 2 L of natural seawater (with the exception of the predator water control, which received 1.5 L seawater + 0.5 L predator water) that had settled for at least 3 days to remove sediment particles. Seawater was supplemented with either Instant Ocean salt or deionized water to reach 20 ppt (± 2 ppt). Each aquarium contained filtered air bubblers for oxygenation. Aquaria were covered with lids to reduce evaporation and stored outside under a covered pavilion in a water bath containing ambient flow-through seawater to regulate temperature. Spat were fed Instant Algae Shellfish Diet 1800 (Reed Mariculture). At the start of the experiment, spat were fed 0.5 mL twice daily, but we increased this amount to 1 mL twice daily as spat grew larger. Complete water changes and aquarium cleanings were performed twice weekly, and predator chemical cues were added to the aquaria immediately after water changes. 

Blue crabs were collected from crab pots near Dauphin Island, AL and stored in the same facility and conditions as described for the predator cue bioassay in 2020 (See related dataset: https://www.bco-dmo.org/dataset/883945). Urine collection methods remained the same as for previous experiments (2020 predator cue bioassay) except urine was pooled from all crabs for this experiment. All crabs for this experiment were fed an oyster diet (i.e., one adult oyster (~6-7 cm in length) twice weekly). Crab were kept in aquaria for a one week acclimation period before beginning a urine extraction regimen. Urine was collected twice weekly from 22 crabs that ranged 13 – 18 cm in size and each crab produced 1.31 ± 0.18 mL. Urine dose concentrations were determined based on the average concentrations of homarine and trigonelline quantified in blue crab urine in the 2020 predator cue bioassay (homarine, 13 ± 21 µM; trigonelline, 3.6 ± 6.9 µM). It was assumed that 1 mL of pooled blue crab urine contained these concentrations, and doses were adjusted by half-log steps until approximated concentrations of homarine and trigonelline spanned four orders of magnitude (Table S3 in Supplemental File PDF). Though initial experimental design was based on an assumed concentration of homarine and trigonelline, all urine doses were reported as urine volume divided by the total volume of seawater within an experimental aquaria. For the six lowest doses, 1 mL aliquots of blue crab urine were prepared via serial dilution. The two highest doses received 5.00 mL and 1.25 mL aliquots of pure urine respectively. All aliquots were frozen at –80 °C to be used on the day of cue addition.

 The preparation and maintenance of the urine dose response experiment was identical to the previous homarine and trigonelline dose response experiment. The experiment was conducted for 56 days (8 weeks) from 08 Aug. 2022 – 03 Oct., 2022 and at the completion of the experiment tile pairs were removed from their jars, measured, and crushed according to previous methodology (Robinson et al. 2014). There were five replicate aquaria per concentration, and within these replicate aquaria, we took an average of 32 crushed oysters. 

Instruments: 
Individual spat width was measured for each crushed oyster to 0.01 mm using a Vernier digital caliper. The force required to crush oysters was measured using a Kistler 5995 charge amplifier and Kistler 9207 force sensor following standard protocol (Robinson et al., 2014). 

Problems/Issues: None.